The present invention relates to the field of lactic acid bacterial starter cultures and in particular there is provided the means of enhancing the growth rate and/or controlling the metabolic activity of lactic acid bacteria by cultivating the lactic acid bacteria in association with a lactic acid bacterial helper organism which has a defeat in its pyruvate metabolism. Such a helper organism is also useful as a means of improving the shelf life and/or quality of edible products.
Lactic acid bacteria are used extensively as starter cultures in the food industry in the manufacture of fermented products including milk products such as e.g. yoghurt and cheese, meat products, bakery products, wine and vegetable products.
As used herein the term xe2x80x9clactic acid bacteriaxe2x80x9d refers to gram-positive, microaerophilic or anaerobic bacteria which ferment sugars with the production of acids including lactic acid as the predominantly produced acid, acetic acid, formic acid and propionic acid. The industrially most useful lactic acid bacteria are found among Lactococcus species, such as Lactococcus lactis, Lactobacillus species, Streptococcus species, Leuconostoc species, Pediococcus species and Propionibacterium species. Also the strict anaerobes belonging to the genus Bifidobacterium is generally included in the group of lactic acid bacteria.
When a lactic acid bacterial starter culture is added to milk or any other edible starting material and appropriate conditions for growth and metabolic activity of the starter culture are provided, the bacteria will start to propagate after a period of time known as the lag phase, during which the bacteria adapt to the new conditions. Once propagation of the bacteria is initiated it is rapid with concomitant conversion of citrate, lactose or other sugars into lactic acid/lactate as the major acidic metabolite, and possibly other acids including acetate, resulting in a pH decrease. In addition, several other metabolites such as e.g. acetaldelyde, xcex1-acetolactate, acetoin, diacetyl and 2,3-butylene glycol (butanediol) are produced during the growth of the lactic acid bacteria.
Generally, the growth rate and the metabolic activity of lactic acid bacterial starter cultures can be controlled by selecting appropriate growth conditions for the strains of the specific starter culture used such as appropriate growth temperature, oxygen tension and content of nutrients. Thus, it is known in the dairy industry that a reduction of the oxygen content of the milk raw material will result in a more rapid growth of the added lactic acid bacteria which in turn results in a more rapid acidification of the inoculated milk. Currently, such a reduction of the oxygen content is carried out by heating the milk in open systems, by deaerating the milk in vacuum or by a sparging treatment. Alternative means of reducing the oxygen content include the addition of oxygen scavenging compounds.
Lactic acid bacterial starter cultures are commonly used in the food industry as mixed strain cultures comprising one or several species. For a number of mixed strain cultures such as yoghurt starter cultures typically comprising strains of Lactobacillus bulgaricus and Streptococcus thermophilus, a symbiotic relationship between the species has been reported, assumingly due to proteolytic activity of at least one of the strains (Rajagopal et al. J. Dairy Sci., 1990 73:894-899). It has also been reported that in such mixed yoghurt cultures, stimulation of growth of the Lactobacillus component is due to the inherent formation of formic acid by the Streptococcus thermophilus (Suzuki et al., 1986). A further example of a symbiotic relationship between strains in a mixed culture is disclosed in EP 0 111 392 A2 where it is demonstrated that selected wild-type Streptococcus thermophilus strains having a relatively high oxygen uptake ability improves the survival of a strictly anaerobic Bifidobacterium species when it is combined with the Streptococcus strain.
However, the prior art is not aware of any generally applicable biological method whereby the growth and metabolic activity of lactic acid bacterial starter cultures can be enhanced. It is therefore a primary objective of the present invention to provide such a method.
Accordingly, the present invention relates in a first aspect to a method of enhancing the growth rate and/or controlling the metabolic activity of a lactic acid bacterial strain, comprising cultivating the strain in association with a lactic acid bacterial helper organism that is defective in its pyruvate metabolism.
In a further aspect there is provided a method of improving the shelf life and/or the quality of an edible product comprising adding to the product a lactic acid bacterial strain that is defective in its pyruvate metabolism and in a still further aspect the invention pertains to a starter culture composition comprising a lactic acid bacterium and a lactic acid bacterial helper organism that in defective in its pyruvate metabolism, said helper organism being capable of enhancing the growth rate and/or controlling the metabolic activity of the lactic acid bacterium.
In yet another aspect the invention pertains to a lactic acid bacterium that is defective in at least one enzyme involved in the pyruvate metabolism and in which a gene capable of regenerating NAD+ is overexpressed.
It is a primary objective of the present invention to provide a generally applicable method of enhancing the growth rate and/or controlling the metabolic activity of a lactic acid bacterial starter culture. The method comprises cultivating the culture in association with a lactic acid bacterial helper organism which is defective in its pyruvate metabolism.
It will be understood that enhancement of the growth rate relates to any effect resulting in a higher number of starter culture cells in the medium after a given period of time, i.e. the lactic acid bacterial cells propagate at a higher rate than that obtained without the helper organism, or the cells start propagating at an earlier point in time at a rate equal to or higher as compared to propagation of the lactic acid bacteria without the helper organism.
As used herein, the expression xe2x80x9ccontrolling the metabolic activityxe2x80x9d refers to the increased or decreased production of any metabolite produced by the starter culture, including the production of acids, such as lactic acid, acetic acid, formic acid and/or propionic acid. Examples of other metabolites of relevance, the production of which may be controlled, include aroma compounds such as acetaldehyde, xcex1-acetolactate, acetoin, diacetyl and 2,3-butylene glycol (butanediol).
In accordance with the invention, the lactic acid bacterial helper organism is defective in its pyruvate metabolism. As used herein the expression xe2x80x9cdefective in its pyruvate metabolismxe2x80x9d indicates that the helper organism in comparison with the parent strain has an altered metabolism of pyruvate, i.e. an increased or decreased production of one or more metabolites derived from pyruvate.
Such an altered metabolism of pyruvate can be the result of the helper organism being defective in its ability to egress at least one enzyme selected from the group consisting of pyruvate formate lyase, pyruvate dehydrogenase, lactate dehydrogenase, acetolactate synthetase, second acetolactate synthetase, acetolactate decarboxylase and diacetyl reductase. As used herein the expression xe2x80x9cdefective in its ability to express any of the above enzymesxe2x80x9d, indicates that the helper organism as compared to the parent strain from which it is derived has a reduced production of the enzyme or that the enzyme is not expressed at all, irrespective of the growth conditions.
Examples of lactic acid bacterial helper organisms which are defective in their ability to express at least one of the above mentioned enzymes include the Lactococcus lactis subspecies lactis strain DN223 which is defective both in the pyruvate formate lyase (Pflxe2x88x92) enzyme and in the lactate dehydrogenase enzyme (Ldhxe2x88x92) and the Lactococcus lactis subspecies lactis strain DN224 which is Ldh defective.
In one useful embodiment of the above method, the cultivation of a lactic acid bacterial strain of the starter culture in association with the helper organism results in an enhancement of the acid production of the strain.
Evidently, the above-mentioned enhanced production of acids will result in a pH decrease of the medium inoculated with the associative culture (a starter culture in association with the helper organism according to the invention) which exceeds that obtained in the same medium inoculated with the starter culture alone. The difference in pH of the medium inoculated with the starter culture alone and the medium inoculated with the associative culture is referred to herein as xcex94pH. In useful embodiments of the invention the enhanced acid production results in a xcex94pH of at least 0.05 after 3 hours or more of cultivation, such as a xcex94pH of at least 0.1 after 3 hours or more of cultivation, e.g. a xcex94pH of at least 0.5 after 3 hours or more of cultivation, such as a xcex94pH of at least 0.8 after 3 hours or more of cultivation, e.g. a xcex94pH of at least 1.0 after 3 hours or more of cultivation.
In useful embodiments of the present invention the lactic acid bacterial starter culture is a mixed strain culture comprising at least two strains of lactic acid bacteria. Examples of such mixed strain cultures are described in the below examples. Thus, in particularly preferred embodiments of the invention the helper organism is capable of enhancing the growth rate of at least one of the mixed strain culture strains and/or capable of controlling the metabolic activity of at least one of the strains of the lactic acid bacterial mixed strain culture. Growth conditions which are in all respects optimal for all strains of such lactic acid bacterial mixed strain cultures may not be found. Therefore, the metabolic activity of a mixed strain culture may be controlled selectively by choosing a temperature which favor an increased production of desired metabolites by one or more strains, but which on the other hand may result in a decreased production of other metabolites by other strains. However, the overall result of cultivating a lactic acid bacterial mixed strain culture with a helper organism according to the invention as compared to the lactic acid bacterial mixed strain culture being cultivated alone is an increased number of cells, an increased production of one or more metabolites, including acids and aroma compounds and/or a decreased production of one or more metabolites.
Industrial production of edible products typically includes process steps such as mixing, pumping or cooling whereby the degree of oxygen saturation of the edible product is increased and, as a result, the edible product starting material may have a relatively high initial oxygen content (high degree of oxygen saturation) which is unfavorable for lactic acid bacterial starter cultures. It has now surprisingly been found that when the starter culture is cultivated in an edible product starting material having an initial degree of oxygen saturation of 10%, or higher such as 20% or higher in association with a helper organism according to the invention, its growth rate is substantially enhanced and/or its metabolic activity is controlled as compared to cultivating it without the helper organism under the same conditions.
In useful embodiments of the invention the helper organism is a lactic acid bacterium capable of reducing the amount of oxygen present in the medium. Thus, in particularly preferred embodiments of the invention the helper organism is capable of reducing the amount of oxygen present in the medium by at least 1% per hour including by at least 10% per hour, such as by at least 20% per hour, e.g. by at least 30% per hour. The reduction may even be by at least 40% per hour including by at least 50% per hour, such as by at least 60% per hour, e.g. by at least 70% per hour, such as by at least 80% or by at least 90% per hour.
The method of enhancing the growth rate and/or controlling the metabolic activity according to the invention implies that an increased growth rate and/or control of metabolic activity of lactic acid bacterial starter culture can be obtained even in a medium having a low degree of oxygen saturation, such as in the range of 1-10%. However, the method may be particularly useful when the lactic acid bacterial starter culture is cultivated in an edible product starting material having an initial oxygen saturation of 10% or more, e.g. 20% or more, such as 40% or more, e.g. 50% or more, such as 60% or more, e.g. 70% or more, such as 80% or more, and e.g 90% or more, such as an initial oxygen saturation of 95% or more.
In general, the helper organism is a derivative of a lactic acid bacterium. As used herein the expression xe2x80x9cderivative of a lactic acid bacteriumxe2x80x9d encompasses a lactic acid bacterial mutant which is derived by selecting a spontaneously occurring mutant of a wild-type strain of a lactic acid bacterium or alternatively, by constructing a mutant of a wild-type lactic acid bacterial strain or a previously mutated strain. This construction can be made by subjecting a strain to any conventional mutagenization treatment including treatment with chemical mutagens and UV light.
A mutant can also be constructed by genetic modifications in the parent strain including deletions, insertions, substitutions of nucleotides. These genetic modifications can be obtained by techniques known in the art to introduce such modificaticons, including DNA recombination techniques including site directed mutagenesis, polymerase chain reaction techniques, random or quasi-random mutagenesis using any mutagen, in vitro mutagenesis or any other method known to introduce genetic modifications into substances comprising or being derived from naturally occurring nucleic acids or amino acids. In accordance with the invention, the derivative of a lactic acid bacterium can e.g. be derived from a Lactococcus species, such as Lactococcus lactis, a Lactobacillus species, a Streptococcus species, a Leuconostoc species, a Pediococcus species, a Propionibacterium species or a Bifidobacterium species.
In this context, one preferred species is Lactocaccus lactis including Lactococcus lactis subspecies lactis including biovar diacetylactis. Examples of suitable helper organisms are Lactococcus lactis subspecies lactis strain DN223 which has been deposited under the accession No. DSM 11036 and Lactococcus lactis subspecies lactis strain DN224 which has been deposited under the accession No. DSM 11037. The DN223 and DN224 strains are described in WO 98/07843. In the following reference examples details are given for the isolation of these strains.
It has been found that derivatives, such as the Move DN223 and DN224 strains, that have, relative to the parents, a reduced production of acid, are particularly suitable in the above method of enhancing growth rate or controlling metabolic activity.
The enhancement of the growth rate and/or controlled metabolic activity obtained by the above method can be provided when cultivating the starter culture in any media supporting the growth of lactic acid bacteria. Thus, the effect can be obtained in a variety of edible product components or ingredients such as milk, meat, flour dough, wine and plant materials, such as vegetables, fruits or fodder crops.
The starter culture and the helper organism is added in amounts which result in a number of viable cells of each component which is at least 103 colony forming units (CPU) per gram of the edible product starting materials, such as at least 104 CFU/g including at least 105 CFU/g, such as at least 106 CFU/g, e.g. at least 107 CFU/g, such as at least 108 CFU/g, e.g at least 109 CPU/g, such as at least 1010 CFU/g, e.g. at least 1011 CFU/g of the edible product starting materials.
In preferred embodiments of the present invention the ratio between helper organism cells and lactic acid bacterial culture cells is in the range of 1000:1 to 1:1000 such as 500:1 to 1:500, e.g. 100:1 to 1:100, such as in the range of 50:1 to 1:50, e.g in the range of 20:1 to 1:20, such as in the range of 10:1 to 1:10 or in the range of 5:1 to 1:5 such as in the range of 2:1 to 1:2.
In the metabolism of lactic acid bacteria it is required to regenerate NAD+. Several of the enzymes involved in the pyruvate metabolism including Ldh is capable of this regeneration by converting pyruvate to lactate. Accordingly, in a lactic acid bacterial strain that has a defect in its pyruvate metabolism which implies that the ability of the strain to regenerate NAD+ is reduced, there is a need for alternative ways of providing the required amount of this essential compound. One such alternative way which is naturally available in lactic acid bacteria is regeneration by means of NADH oxidases of which three types have been reported (Condon, 1987). The first two are non-haem flavoproteins, one of which catalyses the reduction of O2 to H2O2, the other one the reduction of O2 to H2O. One example of the latter type of enzyme, i.e. an H2O forming NADH oxidase is the enzyme encoded by the nox gene. This enzyme regenerates two equivalents of NAD+ under oxygen consumption.
It is therefore contemplated that the enhancing effect of a helper organism according to the invention can be further improved by overexpressing an O2 reducing (i.e. O2 consuming) enzyme including the enzyme encoded by a nox gene present in the organism.
Accordingly, in a further embodiment of the present method the helper organism is one wherein a gene coding for an enzyme that is capable of regenerating NAD+ including the above NADH oxidases is overexpressed. In the present context, the ten xe2x80x9coverexpressedxe2x80x9d indicates that the level of expression of the gene in increased relative to that of the parent strain from which the helper organism overexpressing the gene is derived. Thus, a helper organism that is capable of overexpressing the gene coding for the NAD+ regenerating enzyme preferably expresses the gene at a level which is at least 10% higher than the level at which the gene is expressed in the parent such as at least 25% higher, e.g. at least 50% higher. It is particularly preferred that the level of expression is at least 100% higher than that of the parent.
The overexpression of the gene can be provided by methods which are known in the art such as e.g. by introducing in the helper organism multiple copies of the gene on the chromosome and/or on extrachromosomal elements including plasmids, phages or cosmids.
Alternatively, the overexpression is the result of operably linking a gene or genes naturally occurring in the helper organism or a gene/genes that is/are inserted into the organism to a regulatory sequence that enhances the expression either at the transcriptional or the translational level. In this context, one useful approach is to link the gene operably to a strong homologous or heterologous promoter which optionally is a regulatable promoter. Interesting promoters are tRNA and rRNA promoters including the PI and PII promoters and the purD promoter from Lactococcus lactis subspecies lactis as described in WO 94/16086 to which there is referred.
A regulatable promoter regulating the expression of the gene coding for an NAD+ regenerating enzyme can suitably be regulated by a factor selected from pH, the growth temperature, a temperature shift eliciting the expression of heat chock genes, the composition of the growth medium including the ionic strength/NaCl content and the presence/absence of purine nucleotide precursors, and the growth phase/growth rate of the bacterium.
It is also possible to obtain a helper organism having an increased NAD+ regenerating activity by altering the structure of the enzyme e.g. by modifying the coding sequence or post-translationally by methods which are known per se.
In the present context, an example of a suitable NAD+ regenerating enzyme is the NADH oxidase encoded by the nox gene.
In accordance with the present method, the helper organism capable of overexpressing a NAD+ regenerating enzyme includes an organism wherein the enzyme catalyses the reduction of O2 to H2O or H2O2, e.g. the enzyme having the sequence SEQ ID NO:2 as shown below. In useful embodiments the helper organism is an Ldhxe2x88x92 strain.
As it is described above, lactic acid bacterial strains that are defective in their pyruvate metabolism include strains that are capable of reducing the amount of oxygen in a medium. It has been found that such strains, when used alone, i.e. without the concomitant addition of a starter culture strain, can improve the shelf-life of edible products, Accordingly, it is another objective of the invention to provide a method of improving the shelf life and/or the quality of an edible product, comprising adding to the product a lactic acid bacterial strain that is defective in its pyruvate metabolism as it is defined above. As used herein the term xe2x80x9cshelf lifexe2x80x9d indicates the period of time in which the edible product is acceptable for consumption. In one useful embodiment, the lactic acid bacterial strains is one that has a reduced production of lactic acid including a strain that essentially does not produce lactic acid.
In accordance with the invention, a lactic acid bacterial strain that is useful for improving the shelf-life of edible products includes a strain as described above in which a gene coding for an enzyme that is capable of regenerating NAD30  including the above NADH oxidases is overexpressed.
The above shelf-life improving effect can be obtained in a variety of edible product components or ingredients such as milk including non-pasteurized (raw) milk, meat, flour dough, wine and plant materials, such as vegetables, fruits or fodder crops. As used herein, the term xe2x80x9cmilkxe2x80x9d is intended to mean any type of milk or milk component including e.g. cow""s milk, human milk, buffalo milk, goat""s milk, sheep""s milk, dairy products made from such milk, or whey.
The rate at which the above lactic acid bacterial culture removes oxygen is dependent on the conditions of the medium, e.g. the temperature. With temperatures in the edible product components or ingredients often being lower than room temperature, such as below 10xc2x0 C., e.g. below 5xc2x0 C., the rate of which oxygen is removed may be as low as 1% per hour and still have an impact on the shelf life and/or quality of the edible product.
When used in accordance with the above method the non-acidifying lactic acid bacterial culture is preferably mixed with the edible product at the production site. Thus, as an example, when the edible product is non-pasteurized, raw milk the lactic acid bacterial culture can be added on the dairy farm to the milk subsequent to milking. Conveniently, the culture is added to the fresh milk in a cooling tank at the dairy farm or to a storage tank at a dairy plant.
In accordance with the method of the present invention it is also possible to achieve an enhancement of the biomass yield during starter culture production within a given period of time. Thus, this effect can be obtained when the volume of the starter culture is increased stepwise, which is also referred to in the art as xe2x80x9cbulk starter systemsxe2x80x9d.
As mentioned above, the invention provides in a further aspect a starter culture composition comprising at least one strain of a lactic acid bacterium and a lactic acid bacterial helper organism as described above that is defective in its pyruvate metabolism as also described above, including a helper organism that has a reduced production of lactic acid such as a strain that essentially does not produce lactic acid.
Typically, such compositions comprise the bacteria in a concentrated form including frozen, dried or freeze-dried concentrates typically having a concentration of viable cells which is at least 105 CFU per gram of the composition, such as at least 106 CFU/g including at least 107 CFU/g, e.g. at least 108 CPU/g, e.g. at least 1010 CFU/g, such as at least 1011 CFU/g, e.g. at least 1012 CFU/g, such as at least 1013 CFU/g of the composition. The composition may as further components contain cryoprotectants and/or conventional additives including nutrients such as yeast extract, sugars and vitamins.
In accordance with the invention there is also provided a lactic acid bacterium that is defective in at least one enzyme involved in the pyruvate metabolism as it is described above and in which a gene capable of regenerating NAD+ is overexpressed, including a gene coding for an enzyme catalyzing the reduction of O2 to H2O or H2O2 such as an NADH:H2O oxidase including the enzyme having the sequence SEQ ID NO:2.
As mentioned above, the invention also provides an isolated DNA fragment derived from a lactic acid bacterium comprising a gene coding for a polypeptide having NADH:H2O oxidase activity such as a DNA fragment which is selected from the group consisting of the sequence shown in SEQ NO ID:1 and a variant or derivative hereof which is at least 50% e.g. at least 60% including at least 70% identical with said sequence, and a recombinant DNA molecule comprising such a, DNA fragment. In the present context, the expression xe2x80x9cvariant or derivativexe2x80x9d refers to any modification, including mutations, of the DNA sequence of the above specific sequence including substitution, addition or deletion of one or more nucleotides.